Enzyme and immunohistochemical assessment of myocardial damage after ischaemia and reperfusion in a closed-chest pig model

Abstract

The usefulness of different enzyme and immunohistochemical stains to distinguish reversible and irreversible myocardial cell injury after experimental coronary artery occlusion of varying duration and reperfusion with or without superoxide dismutase as adjunct was investigated. Biopsies or parts of the infarcted and non-infarcted area were rapidly frozen and sectioned in series for enzyme and immunohistochemical evaluation. Sections were stained for the demonstration of phosphorylase, myofibrillar ATPase and mitochondrial oxidative enzymes and also with periodic acid-Schiff, alizarin red S and routine histological stains. Other sections in series were stained with antibodies against fibronectin and the intermediate filament proteins desmin and vimentin. In 49 biopsies a blind quantitative estimation of the area stained for fibronectin, phosphorylase and alizarin red S was performed and evaluated statistically. Phosphorylase, periodic acid-Schiff, fibronectin and alizarin red S allowed delineation of affected myocardium after 30 min of ischaemia followed by reperfusion whereas with the other stains, affected myocardium was readily detectable only after 60 or 90 min of ischaemia followed by reperfusion as well as after 24 h of ischaemia without reperfusion. The immunostaining for fibronectin was very distinct and inversely related to the phosphorylase activity. We show that fibronectin is an excellent marker for damaged cells and that these positively stained myocytes are necrotic as confirmed ultrastructurally. Using alizarin red S as a marker of calcium accumulation in myocytes, a marked discrepancy was observed between the area of fibronectin-containing myocytes and that of myocytes stained by alizarin red S. Calcium accumulation in mitochondria is thus not a prerequisite for myocyte necrosis but does occur only in some of the irreversibly damaged cells. Of special interest is the finding that there was a significant reduction of intracellular calcium in pigs where superoxide dismutase had been used as an adjunct at reperfusion, thus supporting the theory that free radicals do play a role during reperfusion of ischaemic myocardium.